Recombinant nucleoside-specific ribonuclease and method of producing and using same

ABSTRACT

A recombinant ribonuclease is disclosed. The recombinant ribonuclease is produced by introducing a recombinant DNA sequence into a host; activating expression of the recombinant DNA sequence within the host to produce the recombinant ribonuclease; and isolating the recombinant ribonuclease from the host. Additionally, a method of analyzing an RNA sequence includes digesting the RNA with a first recombinant ribonuclease to give digestion products comprising nucleotides of the RNA sequence; and analyzing the digestion products using an analytical method to provide the identity of at least some of the nucleotides. The recombinant ribonuclease includes at least one of a uridine-specific recombinant RNase MC1 and a cytidine-specific recombinant RNase Cusativin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase Application of PCTApplication No. PCT/US2016/029151, filed on Apr. 25, 2016, and is alsorelated and claims priority to U.S. Provisional Patent Application Ser.No. 62/151,546, filed on Apr. 23, 2015, and U.S. Provisional PatentApplication Ser. No. 62/151,640, also filed on Apr. 23, 2015, the entirecontents of which are herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.CHE-1156449 awarded by the National Science Foundation and under GrantNo. GM058843 awarded by the National Institutes of Health. The U.S.Government may have certain rights in this invention.

REFERENCE TO ONE OR MORE SEQUENCE LISTINGS

The accompanying sequence listings, which are incorporated in andconstitute a part of this specification, illustrate embodiments of theinvention and, together with a general description of the inventiongiven above and the detailed description given below, serve to explainthe principles of the invention.

FIELD

The present invention is generally related to the field of recombinantribonucleases, and more particularly, to methods of analyzing an RNAsequence using a recombinant ribonuclease.

BACKGROUND

Nucleoside-specific ribonucleases (hereinafter “RNases”) are importanttools for locating the more than 120 modified nucleosides that may befound in an RNA sequence by the process generally referred to as RNAmodification mapping. RNA modifications may be associated with a varietyof human diseases through both structural and functional roles.Identification and location mapping of nucleoside modifications withinthe overall RNA sequence is important for determining the biologicalroles of nucleoside modifications. Traditionally, RNA-sequencingtechnologies primarily relied on polymerization-dependent copying of RNAinto deoxyribonucleotides through Watson-Crick base pairing. However,this copying leads to a loss of modification information of the originalRNA sequence.

Mass spectrometry (hereinafter “MS”) can directly measure the mass shiftassociated with RNA modifications. One RNA mapping approach involveshydrolysis of a target RNA to yield nucleosides. Then, MS is used tocreate a census of nucleoside modifications and nucleoside-specificRNase digestion of the target RNA is used to identify modificationplacement. Knowledge of the compositional value of one nucleosideresidue imposes a constraint on the number of possible base compositionsfor a given mass value. Thus, to simplify the MS analysis of RNasedigestion products, much effort is expended to determine thecompositional value of at least one nucleoside residue. In practice,base-specific RNase digestion of RNAs followed by separation and MSusing ion-pairing, reverse phase liquid chromatography, or IP-RP-LC-MS,and collision-induced dissociation tandem mass spectrometry, orCID-MS/MS, allows one to map modified nucleosides onto the original RNAsequence.

Few nucleoside-specific or nucleoside-selective RNases are commerciallyavailable. Guanosine-specific RNase T1 and pyrimidine-selective RNase Aare both commercially available and compatible with MS-based RNAmodification mapping. Purine-selective RNase U2 is also commerciallyavailable, but only sparingly so. However, optimal RNA modificationmapping requires the generation of sufficient overlapping digestionproducts from multiple RNases to reduce redundancies in digestionproduct sequences and modification placement.

Alternative strategies for generating overlapping digestion productsexist. These include partial RNase digestion, the use of non-specificnucleases, and alkaline hydrolysis. However, these strategies alsosuffer from certain drawbacks, including non-specificity of digestionand ineffective reaction conditions causing too much or too littledigestion. These drawbacks lead to poor analytical reproducibility andlabor-intensive optimization processes. Therefore, new RNases withcomplementary nucleoside specificity to be used in RNA modificationmapping could prove useful.

RNase MC1, a member of the RNase T2 family first isolated from bittergourd seeds, is known to exhibit uridine-specific cleavage of RNA.Further, cucumber seed derived Cusativin is known to exhibitcytidine-specific cleavage of RNA. However, these RNases are notavailable commercially or on a large scale.

SUMMARY

In an attempt to overcome the noted deficiencies, aspects of the presentinvention are directed toward recombinant RNases.

The present invention is premised on the realization that a codonsequence may be selectively modified to be capable of adjusting usage ofa target gene to resemble that of highly expressed genes, such asribosomal proteins and elongation factors, of a host. Thus,overexpression of desirable non-host genes in a host, such asEscherichia coli (hereinafter “E. coli”), may be enhanced. A firstembodiment of the invention is directed to a recombinant RNase. In anembodiment, the recombinant RNase is a recombinant RNase MC1. In anotherembodiment, the recombinant RNase is a recombinant RNase Cusativin.

A further embodiment of the invention is directed to a method ofanalyzing an RNA sequence. The method includes digesting the RNA with arecombinant ribonuclease to give digestion products comprisingnucleotides of the RNA sequence; and analyzing the digestion productsusing an analytical method to provide the identity of at least some ofthe nucleotides. The recombinant ribonuclease includes at least one of auridine-specific recombinant RNase MC1 and a cytidine-specificrecombinant RNase Cusativin. In one embodiment, the analytical methodmay include high throughput screening. In the same or a differentembodiment, the analytical method may include mass spectrometry.

Yet another embodiment of the invention is directed to a method ofmaking a recombinant RNase. The method includes adding a recombinant DNAsequence into a host; activating expression of the recombinant DNAsequence within the host to produce the recombinant ribonuclease; andisolating the recombinant ribonuclease from the host. The recombinantribonuclease includes at least one of a uridine-specific recombinantRNase MC1 and a cytidine-specific recombinant RNase Cusativin.

The objects and advantages of the present invention will be furtherappreciated in light of the following detailed description and drawingsprovided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with a general description of the invention given above and thedetailed description given below, serve to explain the principles of theinvention.

FIG. 1A provides a listing of all possible codons that are capable ofcoding for the amino acid sequence of RNase MC1.

FIG. 1B provides a nucleotide sequence for a recombinant RNase MC1.

FIG. 2A provides a listing of all possible codons that are capable ofcoding for the amino acid sequence of RNase Cusativin.

FIG. 2B provides a natural nucleotide sequence for RNase Cusativin.

FIG. 2C provides the nucleotide sequence coding for a recombinant RNaseCusativin.

FIG. 2D provides an alignment of the natural nucleotide sequence codingfor RNase Cusativin with the recombinant nucleotide sequence.

FIG. 3 is a comparison of digestion products from RNase T1 and RNaseMC1.

FIG. 4A is a representative growth curve of E. coli cells used toproduce the recombinant RNase MC1.

FIG. 4B is a comparison of the protein amounts purified harvested cellsat various values of OD₆₀₀.

FIG. 4C is an SDS-PAGE of a purified recombinant RNase MC1.

FIG. 4D is a comparison of A₂₆₀ observed with various amounts of RNaseMC1.

FIG. 5 is MS data for the ΨC digestion product.

FIG. 6 is MS data for the UCC digestion product.

FIG. 7 is MS data for the UCCCCCACCACCA digestion product.

FIG. 8 is MS data for the UGGGG[2⁴U] digestion product.

FIG. 9 is MS data for the UCGAAGG[m⁵U] digestion product.

FIG. 10 is MS data for the ΨCGAA digestion product.

FIG. 11 is MS data for the U[Q]UA[ms²i⁶A]A digestion product.

FIG. 12 shows the absorbance at 280 nm of each eluted fraction aftereluting the Cusativin from a CM-Cellulose column.

FIG. 13 shows an SDS-PAGE used to verify the presence of ˜23-25 kDapolypeptide.

FIG. 14 shows the absorbance at 280 nm of each eluted fraction aftereluting the Cusativin from a Sephadex G-75 column.

FIG. 15 is an SDS-PAGE to show the progression of RNase Cusativinpurification.

FIG. 16 shows absorbance when using various protein concentrations afterincubating the samples for two hours at 37° C.

FIG. 17 shows absorbance when using various protein concentrations afterincubating the samples for two hours at 50° C.

FIG. 18-36 show graphical representations of quantitative data for eachdigestion product of Cusativin.

FIG. 37 shows an agarose gel used to monitor the amplification of thesynthetic gene.

FIG. 38 shows an agarose gel used to monitor the presence of recombinantplasmids in E. coli.

FIG. 39 shows an agarose gel used to visualize if recombinant plasmiddigestion had successfully showed the presence of insert.

FIG. 40 shows the SDS-PAGE gel for the column fractions collected fromone colony of Cusativin-producing E. coli.

FIG. 41 shows the SDS-PAGE gel for the column fractions collected fromanother colony of Cusativin-producing E. coli.

FIG. 42 shows absorbance when using various protein concentrations afterincubating the samples for two hours at 37° C.

FIG. 43 shows absorbance when using various protein concentrations afterincubating the samples for two hours at 50° C.

FIG. 44 is an SDS-PAGE to show the relative amount of purified Cusativinfrom two colonies of Cusativin-producing E. coli.

FIG. 45 shows an LC-MS analysis of Cusativin digestion product.

FIG. 46 shows an LC-MS analysis of Cusativin digestion product.

FIG. 47 shows an LC-MS analysis of Cusativin digestion product.

DETAILED DESCRIPTION

Unless clearly defined otherwise from the context, any range of valuespresented in the following Detailed Description and Claims includes eachend point as well as each whole number or fractional part thereof,within the recited range. Additionally, approximating language may beapplied to modify any quantitative representation that may vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term or terms, such as “about” and“substantially,” may not be limited to the precise value specified.

According to exemplary embodiments of the present invention arecombinant RNase is disclosed. The recombinant RNase is prepared from arecombinant nucleic acid sequence. In an embodiment, the RNase is auridine-specific recombinant RNase MC1 having a codon sequence that hasbeen selectively modified to enhance expression in a host. In anembodiment, the RNase MC1 has SEQ ID NO. 1 In another embodiment, theRNase is a cytidine-specific recombinant Cusativin having a codonsequence that has been similarly selectively modified to enhanceexpression in a host. In an embodiment, the recombinant RNase Cusativinhas SEQ ID NO. 2.

Alternative embodiments may include other sequences of RNase MC1 and/orRNase Cusativin wherein the codons are selectively modified to causeenhanced expression in a host. In addition, the host may include anysystem capable of providing the necessary components for proteinexpression. Stated differently, the invention is not limited only to theuse of E. coli as the host.

FIG. 1A provides a listing of all possible codons that are capable ofcoding for the amino acid sequence of RNase MC1, while FIG. 2A providesa listing of all possible codons that are capable of coding for theamino acid sequence of RNase Cusativin. The natural nucleotide sequenceof RNase MC1 is unknown. However, the amino acid sequence is known, andis the basis for the information contained in FIGS. 1A and 1 s providedas SEQ ID NO. 1. A nucleotide sequence for a recombinant RNase MC1 isgiven in FIG. 1B (SEQ ID NO. 2). The natural nucleotide sequence forRNase Cusativin, however, is known and is presented in FIG. 2B (SEQ IDNO. 3). FIG. 2C provides the nucleotide sequence coding for arecombinant RNase Cusativin designed to encourage overexpression in ahost (SEQ ID NO. 4). The amino acid sequence of Cusativin is given bySEQ ID NO. 5. FIG. 2D provides an alignment of the natural nucleotidesequence coding for RNase Cusativin (SEQ ID NO. 3) with the recombinantnucleotide sequence of FIG. 2C coding for the recombinant RNaseCusativin (SEQ ID NO. 4). Although one embodiment each of the nucleotidesequence coding for the recombinant RNase MC1 and RNase Cusativin areprovided, it should be emphasized that, in embodiments of the invention,other recombinant codon combinations could also produce functionalprotein, and the invention is not limited only to the two recombinantsequences provided in FIGS. 1B and 2C.

To prepare the recombinant RNase MC1, a natural MC1 amino acid sequencewas used as a template in a codon modification tool. Restriction siteswere added at the 5′- and 3′-ends to the recombinant codon sequence sothat fusion of a tag for enzyme localization and fusion of apurification tag could later be performed on these available termini.After optimization of expression conditions, active protein wassuccessfully purified and characterized. The uridine-specificity of therecombinant RNase MC1 was confirmed experimentally.

To prepare the recombinant Cusativin, natural Cusativin was purifiedfrom bulk cucumber seeds, and partial protein sequencing was conductedusing MS. This partial protein sequence information was used to identifythe protein coding sequence of the Cusativin gene in the cucumbergenome. A synthetic RNase Cusativin gene with its codons changed toimprove overexpression of the protein in a host was designed. After theexpression conditions for the host was optimized, active protein wassuccessfully purified and characterized. The cytidine-specificity of therecombinant Cusativin was confirmed experimentally.

In embodiments of the invention, the recombinant RNases may be used toanalyze an RNA sequence. To analyze the RNA sequence, the RNA must firstbe digested by the recombinant RNase. Then, an analytical method is usedto analyze the digested RNA.

During digestion of the RNA sequence, without intending to be bound byany particular theory, it is believed that the recombinant RNase MC1cleaves RNA at the 5′-end of the substrate nucleoside, uridine, with thephosphate being retained on the 3′-end of the preceding nucleoside.However, most known RNases cleave RNA at the 3′-end of the substratenucleoside. Indeed, this behavior was observed with the recombinantCusativin, which cleaved RNA at the phosphodiester bond 3′-end with highspecificity.

The analytical method may include one or more of MS, polyacrylamide gelelectrophoresis (hereinafter “PAGE”), and high throughput sequencingmethods, among other methods.

In one embodiment, the digested RNA may be analyzed by MS. For instance,analysis may be performed using IP-RP-HPLC-MS, LC-MS, or CID-MS/MS,among other methods. One of ordinary skill in the art is capable ofselecting the appropriate analytical technique for the particularapplication of the inventive method.

In the same or a different embodiment, the analytical method may be ahigh throughput sequencing method. For instance, the high throughputsequencing method may be Next-Gen RNA-Seq. These methods may be used todiscover potential modifications in any type of cellular RNA, eithercoding or non-coding, including mRNA, tRNA, rRNA, lncRNA, among others.

With an understanding of the cleavage specificity of MC1 and Cusativin,the predicted advantages of including these RNases within a general RNAmodification mapping strategy are significant. For instance, RNase MC1,with its demonstrated uridine specificity, directly complements dataobtained from the guanosine-specific RNase T1, allowing four of the fivenonpseudouridine modifications to be unambiguously placed within theoverall sequence (Table 1). Indeed, a comparison of the sequenced T1digestion products (SEQ ID NO. 6, Seq ID NO. 25) of tRNA^(Tyr I) (SEQID. NO. 7) with that found here after MC1 digestion (SEQ ID NO. 8, SEQID NO. 26, and SEQ ID No. 27)) results in >95% tRNA sequence coverage,as shown in FIG. 3. In FIG. 3, the nucleotides at position 46 and 47,shown in underlined italics, are substituted by CA for the isodecodertRNA^(Tyr II), resulting in a digestion product UCACAGAC instead of UCAand UCGAC from positions 47 to 47:7. Detection of all three digestionproducts indicates the presence of both isodecoders in the sample.Modified nucleosides are denoted by boldface, bracketed text. (Ψ) ispseudouridine, (m⁵U) is 5-methyluridine, (s⁴U) is 4-thiouridine, (Gm) is2′-O-methylguanosine, (Q) is queuosine, (ms²i⁶A) is2-methylthio-N⁶-(3-methyl-2-butenyl)adenosine. Additionally, a furtherdigestion by RNase Cusativin (not shown) provides nearly completesequence coverage.

TABLE 1 MC1 Products T1 Products pGGP (m/z 787.2) UGp UGGGG[s⁴U]p (m/z1012.1) [s⁴U]UCCCGp U[Q]UA[ms²i⁶A]Ap AGp (m/z 1100.5) ΨCp (m/z 628.3)C[Gm]Gp UGCCGp (m/z 811.1) CCAAAGp UCAp (m/z 957.2) CAGp UCGACp (m/z803.1) ACU[Q]UA[ms²i⁶A]AΨCUGp UCGAAGG[m⁵U] (m/z 1320.5) (SEQ ID NO. 10)[Ψ]CGAAp (m/z 815.1) CCGp UCCp (m/z 933.2) UCAUCGp UCCCCCACCACCA (m/z1325.3) ACUUCGp (SEQ ID NO. 9) AAGp [m⁵U][Ψ]CGp AAUCCUUCCCCCACCACCA (SEQID NO. 11)

A further advantage of another nucleoside-specific RNase was noted bythe detection of an MC1 digestion product, UCGCAGACp, m/z 1284.3,positions 46-53, as shown in FIG. 8, by LC-MS that arises from a secondisodecoder (tRNA^(Tyr II)) in the commercial sample. One would predictthat GC-rich RNAs would be more amenable to RNase MC1 analysis as largeroligonucleotide digestion products should be generated. Conversely,GC-poor RNAs would remain amenable to RNase T1, with the two used intandem being a preferred option.

Another significant advantage of RNase MC1 for RNA modification mappingby mass spectrometry is the inherent challenge in characterizingoligonucleotides containing multiple cytidines and uridines. Uridinediffers from cytidine by 1 Da (0 versus NH), and the presence of C-13isotopes, which are readily detected by mass spectrometry, can easilyresult in challenges in differentiating the number and sequence locationof these pyrimidines. This challenge is particularly noteworthy forlarger digestion products wherein the “all light” (C-12) isotope peak isno longer the most abundant. Because digestion with MC1 ensures a singleuridine will be present at the 5′-terminus of each digestion product,the number of cytidines should also be more easily determined based onaccurate mass measurements and prior sequence reconstruction challengeswill be minimized.

The advantages of a uridine-specific nuclease that is inhibited bymodifications (except pseudouridine) should also accrue in other areas.Methylated uridines, for example, should be detectable in RNA-seqanalyses as each site would be missed during MC1 digestion. A comparisonof RNA-seq transcripts generated using this nuclease against the genomicprediction would, therefore, reveal such post-transcriptionalmodifications on a genome-wide basis. Other biochemical approaches thathave effectively been limited to RNase T1, such as RNA footprinting,should also benefit from an additional RNase of high specificity.

Similar advantages are expected to inure to the cytidine-specificCusativin. Additionally, Cusativin exhibits lower rate of cleavage ofthe phosphodiester bond between cytidines. When the RNA has cytidines intandem, Cusativin exhibits a lower rate of cleavage, unlike otherenzymes such as Rnase T1, U2, or MC1. This feature is useful for mappingcytidine modifications even in a cytidine rich sequence of RNA. Such afeature is not known to be present with any of the other RNase.

EXAMPLES

Design of Synthetic Gene and Cloning

Using the MC1 amino acid sequence as a template, a synthetic gene withthe natural codon bias of E. coli was designed using the codonmodification tool from Integrated DNA Technologies available athttp://www.idtdna.com/CodonOpt. The resulting sequence is provided asSEQ ID NO. 1. BamH1 and HindIII sites were added at the 5′- and 3′-ends,respectively, to enable cloning into the IPTG (Isopropylβ-D-1-thiogalactopyranoside)-inducible expression cassette of the pET22bvector (EMD-Millipore). Such a strategy was expected to yield an MC1polypeptide with an N-terminal fusion of the pelB leader peptide and aC-terminal fusion of the His-tag (His)₆ sequence. The pelB signalpeptide is expected to direct the fusion protein to the periplasmicspace, thus obviating any potential deleterious effects of ribonucleaseactivity on host cell RNA machinery.

After confirming the sequence and reading frame of the recombinant clonethrough translation of the experimentally obtained sequence, Rosetta(DE3) cells bearing the recombinant pET22b-MC1 were used for MC1production. Rosetta DE3 cells were transformed with recombinant pET22b(+) MC1 plasmid and plated on an LB-ampicillin (50 μg/mL) andchloramphenicol (34 μg/mL) plate. A single colony was then grownovernight in LB-medium supplemented with ampicillin and chloramphenicolfor a starter culture. The starter culture was subsequently used toinoculate either 0.20 L (small scale) or 1 L media supplemented withantibiotics. The cells were grown in an orbital shaker at 30° C. withconstant shaking at 200 rpm. Expression was induced by adding IPTG tothe broth media.

Optimization of Protein Expression Conditions

Four different experimental variables—growth stage for proteininduction, growth temperature, duration of induction, and concentrationof inducer—were investigated to determine the optimal conditions forinducible expression of RNase MC1.

Specifically, different bacterial growth stages (measured by opticaldensity at 600 nm ranging from 0.3 to 0.9 units) were evaluated forprotein induction. Four different flasks of 200 mL LB media supplementedwith ampicillin and chloramphenicol were inoculated with 2 mL from thesame starter culture of a Rosetta (DE3) cell line bearing therecombinant plasmid. One mL of cells was drawn at regular intervals(30-90 min) to measure the optical density at 600 nm. MC1 expression wasinduced by adding IPTG at different stages in the log-phase growth curveas measured by optical cell densities ranging from 0.3 to 0.9 units atλ₆₀₀. FIG. 4A depicts the representative growth curves followinginduction at each growth stage. In almost all cases, inducibleexpression of MC1 resulted in a change n the growth curve, shiftinggrowth into the log phase. A comparison of the protein amounts purifiedfrom harvested cells revealed higher yields when cells were inducedaround an OD₆₀₀ of 0.6, as shown in FIG. 4B.

The purified protein was analyzed for its relative molecular mass andpurity by SDS-PAGE, as shown in FIG. 4C. This analysis revealed a majorpolypeptide band at M_(r)˜24 kDa with a few minor polypeptides of lowmolecular mass when cells were induced at an OD₆₀₀ of 0.6 and 0.9. Theexpected molecular mass of the MC1-(His)₆ fusion protein (24.1 kDa) issimilar to that observed in SDS-PAGE, suggesting the production of theanticipated polypeptide in the bacterial host. Surprisingly, no suchpolypeptide was observed when cells were induced at OD₆₀₀ of 0.3 and0.7. An examination of the respective growth curves suggests that theoptical density remains essentially unchanged after 3 h of induction atOD₆₀₀ of 0.7, indicating the suspension of metabolic activity in thesehost cells. Induction at early log phase (OD₆₀₀ of 0.3) did not producethe anticipated polypeptide, likely because of a lower cell count andslower multiplication of cultured cells. Although induction at OD₆₀₀ of0.9 resulted in the ˜24 kDa polypeptide, the resulting protein yield wassignificantly less than the amount observed when induction occurred atOD₆₀₀ of 0.6. Hence, induction at OD₆₀₀ of 0.6 was considered optimalfor expression of MC1 protein in the E. coli host.

The optimal duration of MC1 induction was observed to be 2 h from thepoint of IPTG addition at OD₆₀₀ of 0.6, where protein yield peaked at ˜5μg per 200 mL culture. Altering the growth temperature between 30° C.and 37° C. and IPTG concentrations between 0.4 and 1.0 mM had nosignificant impact on protein yield, which remained unchanged at ˜5 μgper 200 mL culture (data not shown).

RNase MC1 Purification

The induced MC1 protein was purified by either a batch process or columnchromatography using a Nickel-Sepharose resin (Novagen) as per themanufacturer's instructions. Batch purification was employed duringinvestigation of the optimal expression conditions, and columnchromatography was performed for large-scale purification. The purifiedprotein yield was measured by Bradford assay. The eluted protein wasexchanged with 100 mM ammonium acetate (pH 5.5) buffer and concentratedusing an Amicon Ultra 0.5 mL filter.

Characterization of MC1 for Mapping Nucleoside Modifications

The presence of putative MC1 protein in the eluted fractions wasconfirmed by the detection of a ˜24 kDa polypeptide on 4%-20% denaturingpolyacrylamide gels (Precise, Thermo Scientific). Nonspecific RNaseactivity of the purified protein was tested by incubating 200 pmol of asubstrate oligonucleotide, UAACUAUAACG (SEQ ID NO. 12), and definedamounts (100-800 ng) of protein at 37° C. for 1 h in a 10 μL volume.UV-absorbance measurements at 260 nm (A₂₆₀) were recorded at T₀ andafter 1 h (T_(1h)) on a nanophotometer (Implen) as per themanufacturer's instructions. Buffer controls containing RNA oligomerwith no protein were also assayed in an identical fashion.

Cleavage of SEQ ID NO. 12 by the RNase will result in oligonucleotideproducts with reduced stacking interactions compared with the startingsubstrate leading to an increase in A₂₆₀ values. Three protein amountswere tested (200, 400, and 800 ng). An increase in A₂₆₀ was measuredwhen increasing the protein amount from 200 to 400 ng, while noadditional increase was detected at 800 ng of protein, as shown in FIG.4D. Presumably, the higher protein amount resulted in no furthercleavage of the oligonucleotide substrate or the increased proteinamount interferes with detection of any additional changes in the UVabsorbance.

The nucleoside-specific enzymatic cleavage of RNA by the purifiedprotein was tested by incubating 3 μg of the commercially available E.coli tRNA^(Tyr I) (Sigma-Aldrich) with a defined amount of purifiedenzyme (100-2000 ng) at 37° C. for 2 h and analyzing the digestionproducts by IP-RP-HPLC-MS. The digestion products were separated on a1×150 mm XBridge C18 column (Waters) employing mobile phase A (200 mMhexafluoroisopropanol [HFIP] [Sigma], 8.15 mM triethylamine [TEA, FisherFair Lawn] in water [Burdick and Jackson, Bridgeport], pH 7.0) andmobile phase B (100 mM HFIP, 4.08 mM TEA in 50% methanol [Burdick andJackson], pH 7.0) at a flow rate of 30 μL/min. The gradient was asfollows: 5% B to 20% B in 5 min; 20% B to 30% B in 2 min; 30% B to 95% Bin 43 min; hold at 95% B for 5 min, followed by equilibration foranother 15 min at 5% B.

The eluted digestion products were subjected to mass analysis using aThermo Scientific LTQ-XL mass spectrometer. The instrument settings forautomatic acquisition of tandem mass spectrum of each mass-selectedprecursor ions are known and will not be further described. The sheathgas, auxiliary gas, and sweep gas at the ionization source were set to25, 14, and 10 arbitrary units (au), respectively. The spray voltage was4 kV, the capillary temperature was 275° C., the capillary voltage was−23 V and the tube lens was set to −80 V.

The theoretical m/z (mass/charge) values of putative digestion products(both U-specific and nonspecific) and the correspondingcollision-induced dissociated (CID) fragment ions were computed usingMongo Oligo (http://mods.rna.albany.edu/masspec/Mongo-Oligo). To confirmuridine specificity for digestion, an LC-MS/MS data set from RNase MC1digestion of E. coli tRNA^(Tyr I) was acquired as described. Each MS/MSspectrum was analyzed for the presence of m/z 328.1 (A), 344.2 (G),304.1 (C), and 305.1 (U) that correspond to the c₁ product ion massesfor the canonical nucleotides. Similarly, the MS/MS spectra were alsoexamined for the presence of m/z 346.2 (A), 362.2 (G), 322.1 (C), and323.1 (U) that correspond to the y₁ product ion masses for the canonicalnucleotides assuming the digestion product was present with a 3′-linearphosphate. If none of these m/z values were observed (as in the case oflonger oligomers or exclusive 2′,3′-cyclic phosphates), the m/z valuescorresponding to the c₂ product ion combinations, e.g.,UA/UG/UC/UU/AU/GU/CU, were considered.

The nucleotide-specific cleavage properties were determined by asystematic examination of the MS/MS spectra from each oligonucleotideprecursor ion whose mass is consistent with a cleavage productcontaining a 3′-phosphate. Evaluation of these data revealedoligonucleotide digestion products exhibiting 5′-uridine residues. Incontrast, the 3′-termini of digestion products were highly variable.Uridine was conspicuous in its absence at the 3′-termini. Two suchrepresentative digestion products, ΨC and UCC, are shown in FIGS. 5A-5Dand 6A-6D, respectively. In FIGS. 5A-5D, (A) is the total ionchromatogram (TIC), (B) is the extracted ion chromatogram (XIC) for m/z628.3, corresponding to the digestion product ΨCp (position 55-56 of SEQID NO. 7), (C) is the mass spectrum associated with the XIC at 11.7min., and (D) is the tandem mass spectrum (MS/MS) of thecollision-induced dissociation (CID) of the m/z 628.3 precursor ion. Theobserved sequence informative product ions, c (with common 5′ end) and y(with common 3′-end) with a subscript denoting the position of cleavageon phosphodiester backbone, are labeled and plotted following thestandard nomenclature of the art. In FIG. 6, (A) is the XIC for m/z933.4, corresponding to the digestion product UCCp (position 55-56 ofSEQ ID NO. 7), (B) is the mass spectrum associated with the XIC at 24.5min, and (C) is the CID tandem mass spectrum of m/z 933.4 precursor ion.As in FIG. 5, the sequence informative fragment ions are labeled.

For longer digestion products where the 5′-terminus could not bedirectly observed in the MS/MS data, fragment ions at the secondposition consistent with the presence of uridine were observed. Based onthis examination of the MS/MS data, a list of m/z values of expectedtRNA^(Tyr I) digestion products and their collision-induced dissociation(CID) fragment ions were calculated assuming cleavage at uridineresidues yielding digestion products with 5′-uridine or 5′-modifieduridine nucleosides.

Using these digestion rules, the entire LC-MS/MS data set fortRNA^(Tyr I) was examined. Among the predicted digestion products wasone from the 3′ end of the tRNA, UCCCCCACCACCA (SEQ ID NO. 9). Thiscytidine-rich digestion product was detected experimentally in highabundance, as shown in FIG. 7. In FIG. 7, (A) is XIC for m/z 1325.1,corresponding to the digestion product SEQ ID NO. 9 (position 73-85 ofSEQ ID NO. 7), (B) is the mass spectrum associated with the XIC at 39.5min, and (C) is the CID tandem mass spectrum of m/z 1325.2 precursorion. Sequence informative fragment ions labeled as in FIG. 5. Anasterisk (*) denotes c-type fragment ions and solid bullet (●) denotesy-type fragment ions. Significantly, no digestion products correspondingto cleavage at cytidine were observed in the LC-MS/MS data indicatingthe specificity of the purified protein toward uridine.

A further examination of the experimental data revealed the presence of3′-linear and 2′,3′-cyclic phosphates for digestion products (arepresentative digestion product is shown in FIG. 6). In FIG. 8, (A) isXICs of m/z 1012.3 (linear phosphate) and m/z 1003.3 (2′,3′-cyclicphosphate), (B) is the MS associated with the XICs at 35 and 33 minutes,and (C) is the CID tandem mass spectrum of m/z 1012.3 precursor ion.Sequence informative fragment ions are labeled. The presence of cyclicphosphates is consistent with an RNase T2 mechanism, which proceeds viathe 2′,3′-cyclic phosphate intermediate before forming the 3′-linearphosphate as the final product. This feature was not enzymeconcentration dependent, as excess enzyme (up to 50× enzyme) did notaffect cyclic phosphate levels. Concentration-independent formation ofthe cyclic phosphate is more consistent with a slow rate ofphosphodiester bond hydrolysis.

Cleavage Preferences at Post-Transcriptionally Modified Uridines

The tRNA^(Tyr I) substrate allowed for an initial investigation into theinfluence of modified nucleosides on the cleavage properties of MC1.This tRNA contains multiple modified nucleosides: 4-thiouridine [s⁴U8],2′-O-methylguanosine [Gm17], queuosine [Q34],2-methylthio-N⁶-isopentenyladenosine [ms²i⁶A37], 5-methyluridine[m⁵U54], and two pseudouridines [Ψ39 and Ψ55]. While uridine andpseudouridine are indistinguishable based on mass, other modificationscan be directly identified by their characteristic mass shift from theunmodified canonical nucleoside, and placed within the overalltRNA^(Tyr I) sequence upon examination of the MS/MS data.

4-Thiouridine and 5-Methyluracil

Of great interest was determining whether MC1 cleavage is impacted bythe presence of modified uridines. As tRNA^(Tyr I) contains threemodified uridines (s⁴U, m⁵U, and Ψ), the LC-MS/MS data were evaluated bygenerating in silico predicted digestion products where s⁴U and m⁵U areeither recognized for cleavage or not and comparing the experimentaldata against these predicted m/z values. The data revealed the presenceof m/z values that correspond to the scenario where s⁴U and m⁵U are notrecognized by MC1. For example, the digestion product UGGGG[s⁴U]p wasdetected at m/z 1012.3 (FIG. 6A,B) and this sequence was confirmed byMS/MS (FIG. 6C). Similarly, m⁵U was not recognized as a substrate forcleavage as noted by detection of the digestion product UCGAAGG[m⁵U] atm/z 1320.5, which also was confirmed by the MS/MS data, as shown in FIG.9. In FIG. 9, (A) is XIC for m/z 1320.2, corresponding to the digestionproduct UCGAAGG[m⁵U] (position 47-54), (B) is the MS associated with theXIC at 37.7 minutes, and (C) is the CID tandem mass spectrum of m/z1320.6 precursor ion. A co-eluting ion (m/z 1284.6) observed in the MScorresponds to the MC1 digestion product (UCACAGAC, position 46-53)belonging to the tRNA^(Tyr II) isodecoder (RY1661). Sequence informativefragment ions are labeled. An asterisk (*) denotes c-type fragment ionsand a solid bullet (●) denotes y-type fragment ions.

If MC1 had recognized these modified nucleosides as substrates, thedigestion products would have been single nucleotides (5′ monophosphatesof s⁴U or m⁵U) as they are followed by either uridine or pseudouridinein the tRNA^(Tyr I) sequence. To confirm that no partial cleavages ats⁴U or m⁵U occurred, the predicted doubly charged digestion productsconsistent with such cleavage, (3)-UGGGG-(7) at m/z 851.1 and(56)-UCGAAGG-(62) at m/z 1160.1, were also searched for within the data.No ions for these m/z values were detected, confirming that s⁴U and m⁵Uare not recognized as substrates by MC1.

Pseudouridine

If MC1 recognizes pseudouridine as a substrate, the expected digestionproducts are predicted at m/z 628.4 (′PC) and m/z 815.1 (ΨCGAA). Theformer was found, as illustrated in FIG. 5, and data consistent with thelatter is shown in FIG. 10. In FIG. 10, (A) is the XIC for m/z 815.4,corresponding to the digestion product ΨCGAAp (position 55-59), (B) isthe MS associated with the XIC at 37.7 minutes, and (C) is the CIDtandem MS of m/z 815.4 precursor ion. Sequence informative fragment ionsare labeled. Although pseudouridine cannot be distinguished from uridineby mass, no other tRNA^(Tyr I) digestion products corresponding to thesequence UC or UCGAA are expected. Therefore, uridine and pseudouridineare indistinguishable in terms of the nucleoside specificity of MC1.

Missed Cleavages

An analysis of the MC1 cleavage patterns of tRNA^(Tyr I) also revealedthat cleavage was not observed if a uridine is preceded by a bulkymodified nucleoside. For example, queuosine at position 34 inhibited MC1cleavage at U35 as noted by digestion products detected at m/z 1100.5and m/z 1091.7, which are consistent with the 3′-linear phosphate and2′,3′-cyclic phosphate digestion products, respectively, for theoligonucleotide U[Q]UA[ms²i⁶A]A, as shown in FIG. 11. Tandem MS of theseprecursor ions confirmed the sequence, revealing the influence of the5′-nucleoside on uridine recognition and cleavage. In FIG. 11, (A) isXIC for m/z 1091.5, (B) is the MS associated with the XIC at 48.3 min,and (C) is the CID tandem MS of m/z 1091.7 precursor ion with sequenceinformative fragment ions labeled. The mass spectrum reveals the doublycharged ions for both linear (m/z 1100.5) and 2′,3′-cyclic phosphate(m/z 1091.7) digestion products of U[Q]UA[ms²i⁶A]A (positions 33-38 ofSEQ ID NO. 7). Fragmentation of queuosine in the oligonucleotide by lossof 115 Da is indicated in the MS.

To determine whether partial digestion at uridines occurs, tRNA^(Tyr I)substrate was incubated with varying amounts of MC1. At lowenzyme/substrate ratios (0.05-1 μg protein per 3 μg of tRNA), partialdigestion at consecutive uridines was noted. These partial digestionscould be eliminated by increasing the enzyme/substrate ratio (2.5 μgprotein per 3 μg of tRNA).

Obtaining and Purifying the Crude Cusativin Protein Extract

Dried seeds of the pickling variety of Cucumis sativus were ground intoflour and extracted overnight at 4° C. using 5 mM sodium phosphate (pH7.2) containing 0.14 M NaCl with vigorous stirring. The extract was thenfiltered through 4 layers of cheesecloth and acidified with 20% glacialacetic acid to pH 4. The filtrate was then centrifuged for 20 min at10,000 rpm, and the pellet was discarded. The supernatant was thencentrifuged for 30 additional min at 15,000 rpm, and the resultantpellet was again discarded. The extract was then filtered throughWhatman No. 1 filter paper to remove the majority of the remainingparticulates.

The filtrate was then applied to a Sephadex G-25 column (2.5 mL bedvolume) equilibrated with 10 mM sodium acetate (pH 4.5). Theflow-through was used for the subsequent step of ion-exchangechromatography.

The eluate was then applied to a CM-Cellulose column equilibrated with10 mM sodium acetate (pH 4.5). The column was then washed with 5 mMsodium phosphate (pH 7), and the eluate was discarded. The protein wasthen eluted from the column with a discontinuous gradient of 5 mM sodiumphosphate (pH 7) containing 50 mM, 100 mM, 200 mM, and 500 mM NaCl. Theeluate was collected in 2 mL fractions, and either Bradford Assay or theUV absorbance at 280 nm was used to identify the fractions containingthe protein.

The presence of protein was not monitored until after the ion exchangechromatography on CM-Cellulose. After eluting the protein fromCM-Cellulose column, the absorbance at 280 nm of each eluted fractionwas monitored, as shown in FIG. 12. The protein-containing ion-exchangechromatography fractions exhibiting highest absorbance were subjected todenaturing gel electrophoresis (SDS-PAGE) to verify the presence of˜23-25 kDa polypeptide, based on the comparison with protein sizestandards (EZ run Rec Protein size standards, Fisher scientific). It wasfound that the target protein (˜25 kDa) eluted mostly in the 5 mM sodiumphosphate containing 200 mM NaCl, as shown in FIG. 13. Additionally,many smaller proteins (5-10 kDa) also eluted in these fractions.

The fractions containing the 25 kDa protein were concentrated to acombined volume of 2 mL or less on a speedvac (Thermo Scientific) andsubjected to size-exclusion chromatography on a Sephadex G-75 column(approximate bed volume of 36 mL, GE Life Sciences) equilibrated with 5mM sodium phosphate (pH 7) containing 500 mM NaCl. The protein waseluted with approximately 1.5 column volumes of 5 mM sodium phosphate(pH 7) containing 500 mM NaCl. 1.5 mL fractions were collected, and aBradford Assay was performed to determine which fractions containedprotein. See FIG. 14. The presence of 23-25 kDa polypeptide in thesize-exclusion column fractions was verified by SDS-PAGE. The targetenzyme eluted after about 1-1.2 column volumes of buffer (fractions30-34) had passed through the column.

The size-exclusion chromatography fractions containing a polypeptide of˜23-25 kDa were pooled, concentrated, and desalted with autoclaved waterusing Amicon Ultra-5 Centrifugal filter devices (size=3 k). Theabsorbance at 280 nm was then used to determine the approximateconcentration of the pure protein (356 μg/mL).

Protein containing aliquots obtained at each step of purification weresubjected to SDS-PAGE to show the progression of RNase Cusativinpurification, as shown in FIG. 15. Following the initial extraction andcentrifugation, the target enzyme (˜23-25 kDa in size) was not visibleon the gel, indicating its presence at very low concentration. Thetarget enzyme was still not visible following gel-filtration on SephadexG-25, as it filters only small molecules. It took ion exchangechromatography on a CM-Cellulose column to concentrate the targetprotein as it eluted with many other smaller proteins in thesefractions. Size exclusion chromatography on Sephadex G-75 with high saltconcentration finally separated the target protein from the smallerproteins and yielded relatively pure enzyme.

Confirmation of Cusativin Activity of Pure Enzyme

Aliquots of the concentrated pure protein (2.5 μL, 5 μL, 7 μL) werecombined with 1 μL 220 mM ammonium acetate and 200 pmol of an RNAoligonucleotide sequence, AUCACCUCCUUUCU (SEQ ID NO. 13). Autoclavedwater was used to dilute the samples to a constant volume of 10 μL.These samples were then incubated for 2 hours at either 37° C. or 50°C., and the absorbance at 260 nm was monitored. Both a blank (autoclavedwater and ammonium acetate) and a negative control (autoclaved water,ammonium acetate, and RNA oligonucleotide) were also incubated. Increasein absorbance at A₂₆₀ was attributed to the presence of active enzyme.

When the general activity assays of the enzyme were initially conductedusing Poly (C) and Poly (U), no increase in absorbance was observedafter incubation. When activity assays were conducted using the RNAoligonucleotide with the purified (more concentrated) protein, as shownin FIG. 15, significant increases in absorbance were observed whencompared to the negative control (0 μL protein), as shown in FIGS. 16and 17. These increases in absorbance were observed after incubating thesamples for two hours both at 37° C. and 50° C. Initial analysis of thedigested RNA on LC-MS did not show any digestion products; however, whendiluted enzyme was used, digestion products were observed.

Several concentrations of the purified enzyme were incubated with E.coli tRNA^(Tyr) (SEQ ID NO. 7) for 1-2 hours at either 37° C. or 50° C.One μL (˜35.6 ng), 2 μL (˜71.2 ng), or 5 μL (178 ng) of diluted enzyme(1 μL stock enzyme diluted into 10 μL with water) and 1 μL of undilutedenzyme (356 ng) were incubated with tRNA^(Tyr) at both temperatures. ThetRNA^(Tyr) digestion products were then analyzed by IP-RP-LC-MS/MS.Mass-to-charge (m/z) values of theoretically expected digestion productsbased on assumed cytidine-specific cleavage of RNA and their productions following CID of digestion product precursor ions were computed byMongo Oligo Mass Calculator(http://mods.rna.albany.edu/masspec/Mongo-Oligo).

Because the modified nucleotide sequence of tRNA^(Tyr) is known, thetheoretical digestion products of tRNA^(Tyr) were compiled for CpN bondcleavage, and the raw MS data was initially searched for the respectivem/z of digestion product precursor ions. The nucleotide sequence ofthese precursor ions was further confirmed by CID where the presence ofsequence-informative product ions was scored. Additionally, digestionproducts resulting from cleavages at other nucleobases were alsoassessed to evaluate the specific cleavage of RNA by Cusativin enzyme.This was necessary because of the known occasional cleavage of RNA atcertain uridines by Cusativin. The digestion products found in thisstudy following incubation of tRNA^(Tyr) with 1 μL of the dilutedprotein (˜36 ng) are shown in Table 2. Each of these digestion productsfound with 36 ng of enzyme was also searched for their presence in othersamples, where different amounts of purified protein were used. The ionchromatogram peaks for each of the digestion product precursor ions wereintegrated using Xcalibur software. Graphical representations of thisquantitative data for each digestion product are shown in FIGS. 18-36.3′-linear phosphates were found on all of the cytidine-specificproducts, while only 3′-cyclic phosphates were found on the uridinespecific products.

TABLE 2 C-specific - C-specific - U-specific - No Missed Missed LowCleavages Cleavages Abundance GAGC GGUGGGG[s⁴U]UCCC GGU 3′ > p (SEQ IDNO. 14) AAAGGGAGC [Gm]GCC AGACU 3′ > p AGAC UGCC GU 3′ > pU[Q]UA[ms²i⁶A]A[Ψ]C GAAUCC UGC/GUC UUCCCCC GUC ACC AUC ACCA GACUUCCCCCACCACCA (SEQ ID NO. 15) UUC GAAGG[m⁵U][Ψ]C

Design of Synthetic Gene, Amplification, and Purification

The purified protein was digested with Trypsin. The tryptic peptidesidentified by LC-MS analysis of Cusativin following treatment withproteolytic enzyme trypsin are shown in Table 3. Tryptic peptides fortwo other small seed coat proteins were also found. The first twopeptides listed in the table were previously known in the literature.The amino acid sequences of a portion of the tryptic peptides wereblasted against Cucumis sativus protein database(GCF_0000040752_ASM407V2_protein.faa) using Protein Lynx (Waters) toidentify the predicted polypeptide as an RNase MC-like protein.

TABLE 3 Tryptic Peptides Tryptic Peptides in Our Study from LiteratureSFTIHGLWPQK SFTIHGLWPNK (SEQ ID NO. 16) (SEQ ID NO. 16) YFQTAINMRYFQTAINMR (SEQ ID NO. 17) (SEQ ID NO. 17) HGIDLLSVLR PPXGHEXNK (SEQ IDNO. 19) (SEQ ID NO. 18) IAHLENDLNVVWPNVVTGNNK (SEQ ID NO. 20) YVGR (SEQID NO. 21) ASNGQVLLTEIVMXFDDDXXTL (SEQ ID NO. 22)

A conserved domain architecture search on the NCBI database shows thatCusativin is a T2-type RNase. Similar results were obtained through theuse of InterPro (http://www.ebi.ac.uk/interpro/).

A synthetic gene with modified codons was designed based on theidentified amino acid sequence so as to enable enhanced proteinexpression in E. coli, as provided in SEQ ID NO. 4. Restriction siteswere added to both ends to enable cloning into the protein expressionvector, pET 22b (Novagen). The synthetic DNA (651 bp) was made as geneblock by IDT DNA technologies.

The gene was amplified via PCR. 5 μL 10×PCR buffer, 1 μL dNTPs, 0.5 μLsynthetic DNA template, and 0.6 μL PfuTurbo DNA Polymerase werecombined. Additionally, to the mixture were added 0.5 μL each of theforward and reverse primers, ATGGAAAAATGGAAAAGACCAAAAGTGTCGATG (SEQ IDNO. 23), and AAAAATAAATGAGCCTGCGCAATTGG (SEQ ID NO. 24), respectively,which also have sequences for BglII and HindIII for restrictionendonucleases at 5′ and 3′-ends, respectively. The primer concentrationwas 100 pmol/μL. These primers enabled easy amplification of the genesequence in this exemplary system, but other primers may be used inother embodiments of this invention. The resulting mixture was dilutedwith water to a total volume of 50 μL. A total of three samples wereprepared and subjected to the following PCR cycle: 94° C. for 2 minutes,92° C. for 0.5 min, 35 cycles of 57° C. for 0.5 min and 72° C. for 2min, and one cycle of 72° C. for 5 min Agarose gel (1.0%)electrophoresis was performed to verify the size of the PCR product.

The amplified gene was purified using the QIAquick PCR Purification Kit(spin protocol). A 1.0% agarose gel was used to check the success of thepurification, as shown in FIGS. 37 and 38.

The gene was digested using restriction enzymes BglII and HindIII. Thefollowing 50 μL reaction mixture was incubated at 37° C. for 2 hours: 15μL DNA, 5 μL NEB 2.1 Buffer, 1.5 μL BglII, 1 μL HindIII. Additionally, asecond experiment was performed using 10 μL DNA. The digested DNA waspurified using the QIAquick PCR Purification Kit (spin protocol).

The purified gene was ligated into a pET-22b(+) vector. The vector hadpreviously been digested with BamHI and HindIII. The ligation reactionmixture was as follows: 9.5 μL DI water, 2 μL 10× ligase buffer, 2 μLpET-22b(+) vector, 6 μl of the digested gene, and 0.5 μL of ligase. Thisreaction mixture was kept at room temperature for 1 hour, and thenchilled at 4° C. for about 5 days.

The ligation mixture was then transformed into BL21 E. coli cells. 10 μLof the ligation mixture was added to two different 1.5 mL tubes of theBL21 E. coli cells and vortexed. The tubes were then put on ice for 30minutes, incubated at 42° C. for 2 min, and then put back on ice for 2min 200 μL SOC media was added to each tube, which were then incubatedand agitated at 37° C. for 1 hour and 10 minutes. The transformed cellswere then plated on LB+amp plates. The plates were then allowed to sitat room temperature for 15 min prior to being placed in the 37° C.incubator overnight.

Following overnight incubation, 10 colonies were collected from theplate. The colonies were collected by stabbing an autoclaved pipet tipinto the colony. This tip was then rubbed onto the bottom of a PCR tubeand then dropped into a test tube containing 1 mL of LB media with 0.05μg/mL ampicillin. The test tubes were incubated overnight at 250 rpm andat 37° C. 38 μL of PCR mixture (similar to the mixture used foramplification of the gene, with the colony substituted as the template)was added to each PCR tube. The colonies were then subjected to thefollowing PCR cycle: 2 min at 94° C., 0.5 min at 92° C., 30 cycles of0.5 min at 57° C. followed by 2 min at 72° C., and finally 5 min at 72°C. A 1.0% agarose gel of the PCR products was performed to visualizewhich colonies contained the gene.

To the colonies containing the gene were added 10 mL of LB mediasupplemented with 0.05 mg/mL ampicillin. These cultures were incubatedat 37° C. on a shaker (Innova) overnight. Experiments were conductedwith other media, including terrific broth, and autoinduction was alsotried.

0.5 mL of each colony was placed in 0.5 mL 65% glycerol and put in thedeepfreeze to serve as future culture stock. The remaining cultures ofeach colony were pelleted. Pelleting was performed using 1.5 mLEppendorf tubes centrifuged for 1 min at 6700 rpm. The supernatant wasthen discarded. Additional cell culture was added to the tube and spundown. This pelleting procedure was repeated until all of the cellculture had been pelleted. The plasmids were purified from the pelletsusing a Qiagen miniprep kit.

The purified plasmids were then digested using the following recipe: 5μL plasmid, 5 μL NEB buffer 2.1, 1.5 μL BglII, 1 μL HindIII, and 37.5 μLwater. The reaction mixtures were incubated for two hours at 37° C. andrun on a 1.0% agarose gel to visualize if the digest had beensuccessful, as shown in FIG. 39.

The purified plasmids were then prepared for sequencing. The sequencingdata confirmed that the purified plasmids were the pET-22b(+) vectorwith the inserted Cusativin gene. The stock colonies (in the deepfreeze)containing these correct plasmids were then cultured in 500 mL LB mediacontaining 0.05 mg/mL ampicillin. The cells were induced to produce therecombinant enzyme by adding IPTG to the culture. His-tag columnchromatography was used to purify the protein from the cells followingoverexpression. Table 4 shows the protein content in each elutionfraction as determined by Bradford Assay. Based on this data it wasfound that each 500 mL culture yielded approximately 34 μg of targetprotein total in the three elution fractions combined. FIGS. 40 and 41show the SDS-PAGE gels for the column fractions collected from eachclone (at least 5 μg protein were loaded in each lane). All threeelution fractions in each purification were found to contain targetprotein, so they were all concentrated and used for activity assays.

TABLE 4 Fraction # (Total Volume) Clone 4 Clone 9 Fraction 1 (500 μL)0.5 μg in 50 μL 0.5 μg in 50 μL Fraction 2 (950 μL) 1 μg in 50 μL 1 μgin 50 μL Fraction 3 (500 μL) 1 μg in 50 μL 1 μg in 50 μL

To prepare the lysate, 2 mg of lysozyme was added for each mL of cellsuspension. The suspensions were then chilled at 4° C. for 30 min.Following incubation at 4° C., the suspensions were centrifuged for 30min at 15000 rpm. The supernatant was then filtered through 0.45 μmDurapore filters. The filtered supernatant was loaded onto the chargednickel column three times. The column was then washed with 10 columnvolumes 1× bind buffer and then washed with 5 column volumes 1× washbuffer. The recombinant enzyme was eluted in three fractions. The firstfraction was 500 μL elute buffer, the second fraction was 750 μL elutebuffer (loaded on column twice) plus an additional 200 μL elute buffer,and the third fraction was 500 μL elute buffer. The column was thenstripped with 6 column volumes 1× strip buffer. Bradford Assay was usedto determine the protein concentration of each fraction, and at least 5μg of protein for each fraction were analyzed by SDS-PAGE to check forpresence of the 23-25 kDa protein. Protein elution fractions from eachclone were concentrated and desalted with water using Amicon Ultra-5Centrifugal filter devices (size=3 k).

Characterization of Recombinant Cusativin for Mapping Nucleoside

Modifications

The activity of the enzyme was tested by incubating aliquots of proteinwith RNA and checking the A₂₆₀. The activity assay was performed using 1μL of a 1:10 dilution of the concentrated protein, 1 μL concentratedprotein, 2 μL concentrated protein, and 5 μL concentrated protein. Eachsample had a total volume of 10 μL and contained 200 pmol RNAoligonucleotide sequence, AUCACCUCCUUUCU (SEQ ID NO. 13), and 1 μL 220mM ammonium acetate. The blank contained no protein or RNA, and thenegative control contained no protein. The samples were incubated atboth 37° C. and 50° C., and the absorbance was checked using theNanodrop.

1 μL concentrated protein and 1 μL of a 1:10 protein dilution did notshow any increase in A₂₆₀ following incubation with RNA oligonucleotide.However, when 2 μL (0.02 μg) and 5 μL (0.1 μg) of concentrated proteinwere used, the absorbance did increase. These increases in absorbanceare shown in FIGS. 42 and 43.

SDS-PAGE was used to approximate the amount of protein contained in theconcentrated fractions. One gel was run with 1 μL and 5 μL of theprotein concentrated from each clone. Another gel was run with 20 μL ofthe protein from each clone. Each gel also had lanes where 0.1 μg BSA,0.5 μg BSA, 1 μg BSA, 3 μg BSA, and 6 μg BSA were run. As shown in FIG.44, the concentrated clone 9 elution fractions contained approximately0.2 μg protein in 20 μL, while the concentrated clone 4 elutionfractions contained much less protein. Each concentrated protein samplehad a total volume of approximately 100 μL.

The RNA digestions were prepared by placing 3 μL tRNA^(Tyr) into anEppendorf tube for each digestion. The tRNA was then incubated at 95° C.for 2 min, followed by 2 min at 4° C. Then, 10 μL of 220 mM ammoniumacetate and a designated amount of protein (5 μL concentrated, 1 μL 1:5dilution, 1 μL 1:10 dilution, and 1 μL 1:20 dilution) were added to eachsample. The samples were then incubated for 2 hours and dried in theSpeedVac.

As shown in FIGS. 42 and 43, the recombinant enzyme did exhibit RNaseactivity. The slight increases in A₂₆₀ observed are very similar to theactivity observed with the enzyme purified from the seeds (FIGS. 16 and17).

FIG. 45 shows an LC-MS analysis of Cusativin digestion product,U[Q]UA[ms²i⁶A]A[Ψ]C, from tRNA^(Tyr). The recombinant protein (0.1 μg)was used to digest E. coli tRNATyr (2 μg) and analyzed by LC-MS. In FIG.45, (A) is the TIC of all the digestion product precursor ions observedin the analysis, (B) is the XIC for m/z 1406.4, corresponding to thelinear phosphate of U[Q]UA[ms²i⁶A]A[Ψ]C, (C) is the XIC for m/z 1397.4,corresponding to the 2′,3′-cyclic phosphate, (D) is an MS depicting themultiply charged ions, and (E) is the CID-based sequencing ofU[Q]UA[ms²i⁶A]A[Ψ]C with a 3′-phosphate. Sequence informative productions (c_(n) having a common 5′-end and y_(n), having a common 3′-end)from which the sequence is reconstructed are labeled on the spectrum.

Additionally, Cusativin cleaves RNA at modified cytidines unlike RNaseMC1. FIG. 46 provides the LC-MS analysis of Cusativin digestion product,UGGAA[m⁷G]UC[m5C]>p, from yeast tRNA^(Phe). In FIG. 46, (A) is the TIC,(B) is the XIC for m/z 1479.4, corresponding to cyclic phosphate, (C) isthe MS corresponding to peak retention time at 32.94 min depicting thedoubly charged ions, and (D) is the CID-based sequencing ofUGGAA[m⁷G]UC[m5C] with a 2′-3′ cyclic phosphate. A portion of theobserved sequence informative product ions (c_(n) having a common 5′-endand y_(n) having a common 3′-end) are shown in the spectrum. Thecharacteristic product ion that corresponds to the loss of [m⁷G] fromthe molecular ion, which is at the highest abundance (m/z 1396.2), isshown.

Although Cusativin exhibits cytidine-specific cleavage of RNA, Cusativinexhibits a lower rate of cleaveage of the phosphodiester bond betweenmultiple cytidines in tandem, as shown in FIG. 47. FIG. 47 shows anLC-MS analysis of Cusativin digestion product, UUCCCCC>p, from yeasttRNA^(Phe). (A) is the TIC, (B) is the XIC for m/z 1067.8, correspondingto cyclic phosphate, (C) is the MS corresponding to peak retention timeat 35.9 min depicting the doubly charged ions, and (E) is the CID-basedsequencing of UUCCCCC>p. A portion of the observed sequence informativeproduct ions (c_(n) with a common 5′-end and y_(n) with a common 3′-end)are shown in the spectrum.

This has been a description of the present invention along with thevarious methods of practicing the present invention. However, theinvention itself should only be defined by the appended claims.

What is claimed is:
 1. A recombinant ribonuclease selected from the group consisting of a uridine-specific recombinant RNase MC1 encoded by the nucleotide sequence of SEQ ID NO. 2 and a cytidine-specific recombinant RNase Cusativin encoded by the nucleotide sequence of SEQ ID NO.
 4. 2. (canceled)
 3. The recombinant ribonuclease of claim 1 wherein the recombinant ribonuclease is encoded by the nucleotide sequence of SEQ ID NO.
 2. 4. The recombinant ribonuclease of claim 1 wherein the recombinant ribonuclease is encoded by the nucleotide sequence of SEQ ID NO.
 4. 5. A method of analyzing an RNA sequence comprising: digesting the RNA with a first recombinant ribonuclease selected from the group consisting of a uridine-specific recombinant RNase MC1 encoded by the nucleotide sequence of SEQ ID NO. 2 and a cytidine-specific recombinant RNase Cusativin encoded by the nucleotide sequence of SEQ ID NO. 4 to give digestion products comprising nucleotides of the RNA sequence; and analyzing the digestion products using an analytical method to provide the identity of at least some of the nucleotides.
 6. The method of claim 5 further comprising digesting the RNA with a second ribonuclease.
 7. The method of claim 6 wherein the second ribonuclease is selected from the group consisting of RNase T1, RNase A, RNase U2, and mixtures thereof.
 8. (canceled)
 9. The method of claim 5 wherein the first recombinant ribonuclease is a uridine-specific recombinant RNase MC1 encoded by the nucleotide sequence of SEQ ID NO.
 2. 10. The method of claim 5 wherein the first recombinant ribonuclease is a cytidine-specific recombinant RNase Cusativin encoded by the nucleotide sequence of SEQ ID NO.
 2. 11. The method of claim 5 wherein the analytical method is selected from the group consisting of mass spectrometry, polyacrylamide gel electrophoresis, a high throughput sequencing method, and combinations thereof.
 12. The method of claim 11 wherein the analytical method includes mass spectrometry.
 13. The method of claim 11 wherein the analytical method includes a high throughput sequencing method.
 14. The method of claim 13 wherein the high throughput sequencing method includes RNA-Seq.
 15. The method of claim 5 wherein the RNA is an mRNA, tRNA, rRNA, IncRNA, or a combination thereof.
 16. A method of making a recombinant ribonuclease selected from the group consisting of a uridine-specific recombinant RNase MC1 encoded by the nucleotide sequence of SEQ ID NO. 2 and a cytidine-specific recombinant RNase Cusativin encoded by the nucleotide sequence of SEQ ID NO. 4 comprising: introducing a recombinant DNA sequence into a host; activating expression of the recombinant DNA sequence within the host to produce the recombinant ribonuclease, wherein the recombinant ribonuclease includes at least one of a uridine-specific recombinant RNase MC1 and a cytidine-specific recombinant RNase Cusativin; and isolating the recombinant ribonuclease from the host.
 17. The method of claim 16 wherein the host is E. coli.
 18. (canceled)
 19. The method of claim 16 wherein the recombinant ribonuclease is a uridine-specific recombinant RNase MC1.
 20. The method of claim 16 wherein the recombinant ribonuclease is a cytidine-specific recombinant RNase Cusativin. 